The overall objective of the proposed research is to understand the mechanisms of animal sensitivity and resistance to 2,3, 7, 8- tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAH), especially resistance developing after long-term (multi-generation) exposure associated with hazardous waste sites. Differential sensitivity to the lethal, reproductive, immunotoxic, and carcinogenic effects of PHAH occurs in species, population, strains, sexes, and developmental stages of animals. The genetic and epigenetic mechanisms underlying differential PHAH sensitivity in natural populations are unknown. Most of the biological activity of PHAH is mediated through binding to the aromatic hydrocarbon receptor (AhR), a ligand-activated transcription factor that controls the expression of genes involved in biotransformation and in cell growth and differentiation. The expression and function of Ah receptors and other components of the AhR-dependent signal transduction pathway in specific cell types are likely to be critical in determining sensitivity of species, populations, and subpopulations to PHAH exposure. Studies to address these issues will be conduct using the teleost Fundulus heteroclitus, which has been shown to develop TCDD resistance and possesses a number of other advantages as a vertebrate model for chemical resistance. Specific aims of the project include; 1) Examination of differential PHAH sensitivity/resistance in populations of F. heteroclitus inhabiting new Bedford harbor and other sites. The resistance and its heritability will be characterized i adult fish sampled from the environment and their progeny raised in the laboratory. 2) The hypothesis that alterations in the AhR-dependent signal transduction pathway underlie the development of resistance will be addressed by examining key steps in the pathway between receptor binding and changes in gene expression in the liver of sensitive and resistant adult fish. Parameters to be measured will include the expression of the AhR and Ah receptor nuclear translocator (ARNT) proteins and their function (ligand binding, transformation, DNA binding). 3). Potential extrahepatic sites of altered AhR and ARNT expression and function will be evaluated in sensitive and resistant fish, focusing on endothelial cells, as these appear to be primary targets for AhR agonists. 4) Endothelial cell structure and function and possible alterations in the AhR-dependent signal transduction pathway will be examined in heart tissue of sensitive and resistant fish treated with TCDD. 5) Responses to PHAH will be examined in primary cultures of hepatocytes and endothelial cells from sensitive and resistant fish. The proposed field- and laboratory-based studies will provide a link between biomedical and ecological aspects of research on the impact of superfund sites, enhancing the development of fish as sentinel species and enhancing the identification of vertebrate markers of genetic susceptibility associated with increased health risks in populations and subpopulations exposed to toxic waste.